Glutamate is the predominant excitatory neurotransmitter in the mammalian central nervous system. An extracellular accumulation of glutamate causes excessive activation of glutamate receptors and cell death through excitotoxic mechanisms. Unlike other classical neurotransmitters, that are recycled directly into the presynaptic nerve terminal, most glutamate is cleared by two astroglial glutamate transporters, called GLAST and GLT-1 (or EAAT1 and EAAT2). These transporters maintain very low synaptic concentrations of glutamate, estimated at ~25 nM, in an environment that contains millimolar concentrations of glutamate. These transporters are enriched on the fine processes of astrocytes that sheath synapses. We recently developed physical evidence (co-immunoprecipitation, mass spectrometry, reverse immunoprecipitations) and anatomic evidence (co-localization in individual astrocytes in organotypic slice cultures) that these transporters exst in a complex with the Na+/K+ ATPase, most of the enzymes in glycolysis, and mitochondria. This complex is observed in fine processes of astroglia. In the first aim, we will identify specifi domains of GLT-1 and GLAST that support the interactions/co- compartmentalization. We wish to identify potential scaffolding proteins that may form a linkage between the transporters and mitochondria. As has been observed with mitochondria at synapses or at nodes of Ranvier, we propose that neural activity recruits mitochondria to regions where transporters are enriched. In the second aim, we will study the effects of neuronal activity on this co-compartmentalization and define the mechanisms involved. Finally, we will test the hypothesis that formation of these complexes is required for glutamate-dependent changes in glycolysis and a shift in glutamate metabolism (from conversion to glutamine to glutamate oxidation). Compartmentalization of the astroglial glutamate transporters with these proteins and mitochondria provides an opportunity to spatially match energy production and buffering capacity. It also has implications for disposition of the glutamate. Therefore, our proposed research will impact our understanding of fundamental aspects of glutamate handling and metabolism. They will also define a novel molecular mechanism that matches astroglial energetic demands to changes in neuronal activity. PUBLIC HEALTH RELEVANCE: For approximately two decades, it has been clear that failure to clear glutamate contributes to the brain damage observed in stroke, neurodevelopmental disorders, and neurodegenerative diseases. This failure to clear glutamate occurs, at least in part, because of impaired energy mobilization. Our long-term goal is to understand how extracellular glutamate is controlled so that it will be possible to intervene and prevent the debilitating effects of these disorders.